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nl1 orf  (Addgene inc)


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    Addgene inc nl1 orf
    ( A ) Schematic depicting inducible release constructs. Multiple copies of self-associating F M domains were fused to target proteins downstream of an ER signal peptide and upstream of a fluorescent protein (FP). DDS dissociates F M domains allowing synchronous ER exit. A furin cleavage site allows removal of the F M domains as they transit the GA. A thrombin cleavage site was included in some constructs so that the FP could be selectively removed from proteins localized at the PM. ( B ) Comparison of 3xF M -mEOS-GluA1 with the endogenous ER-marker BiP before release (left panel) and the Golgi marker GM130 1 hr after DDS addition (right panel). ( C ) Detection of GluA1 surface delivery at various time points following addition of DDS by surface labelling against the extracellular HA-tag of 3xF M -GluA1. ( D ) Quantification of GluA1 surface delivery shown in C (mean ± SEM, n = 10–12 neurons/timepoint from 2 independent experiments). All values normalized to neurons that were not treated with DDS. ( E ) Time-course of <t>NL1</t> surface delivery (mean ± SEM, n = 9–10 neurons/timepoint from 2 independent experiments). ( F ) Localization of surface GluA1 after ER-release. Images taken from insets in panel C. Scale bar, 2 µm.
    Nl1 Orf, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/nl1 orf/product/Addgene inc
    Average 93 stars, based on 13 article reviews
    nl1 orf - by Bioz Stars, 2026-03
    93/100 stars

    Images

    1) Product Images from "Golgi-independent secretory trafficking through recycling endosomes in neuronal dendrites and spines"

    Article Title: Golgi-independent secretory trafficking through recycling endosomes in neuronal dendrites and spines

    Journal: eLife

    doi: 10.7554/eLife.27362

    ( A ) Schematic depicting inducible release constructs. Multiple copies of self-associating F M domains were fused to target proteins downstream of an ER signal peptide and upstream of a fluorescent protein (FP). DDS dissociates F M domains allowing synchronous ER exit. A furin cleavage site allows removal of the F M domains as they transit the GA. A thrombin cleavage site was included in some constructs so that the FP could be selectively removed from proteins localized at the PM. ( B ) Comparison of 3xF M -mEOS-GluA1 with the endogenous ER-marker BiP before release (left panel) and the Golgi marker GM130 1 hr after DDS addition (right panel). ( C ) Detection of GluA1 surface delivery at various time points following addition of DDS by surface labelling against the extracellular HA-tag of 3xF M -GluA1. ( D ) Quantification of GluA1 surface delivery shown in C (mean ± SEM, n = 10–12 neurons/timepoint from 2 independent experiments). All values normalized to neurons that were not treated with DDS. ( E ) Time-course of NL1 surface delivery (mean ± SEM, n = 9–10 neurons/timepoint from 2 independent experiments). ( F ) Localization of surface GluA1 after ER-release. Images taken from insets in panel C. Scale bar, 2 µm.
    Figure Legend Snippet: ( A ) Schematic depicting inducible release constructs. Multiple copies of self-associating F M domains were fused to target proteins downstream of an ER signal peptide and upstream of a fluorescent protein (FP). DDS dissociates F M domains allowing synchronous ER exit. A furin cleavage site allows removal of the F M domains as they transit the GA. A thrombin cleavage site was included in some constructs so that the FP could be selectively removed from proteins localized at the PM. ( B ) Comparison of 3xF M -mEOS-GluA1 with the endogenous ER-marker BiP before release (left panel) and the Golgi marker GM130 1 hr after DDS addition (right panel). ( C ) Detection of GluA1 surface delivery at various time points following addition of DDS by surface labelling against the extracellular HA-tag of 3xF M -GluA1. ( D ) Quantification of GluA1 surface delivery shown in C (mean ± SEM, n = 10–12 neurons/timepoint from 2 independent experiments). All values normalized to neurons that were not treated with DDS. ( E ) Time-course of NL1 surface delivery (mean ± SEM, n = 9–10 neurons/timepoint from 2 independent experiments). ( F ) Localization of surface GluA1 after ER-release. Images taken from insets in panel C. Scale bar, 2 µm.

    Techniques Used: Construct, Comparison, Marker

    ER-retained 4xF M -SEP-NL1 coexpressed with an engineered ER-marker TfR-mCh-KDEL in a COS7 cell. Scale bar, 5 µm.
    Figure Legend Snippet: ER-retained 4xF M -SEP-NL1 coexpressed with an engineered ER-marker TfR-mCh-KDEL in a COS7 cell. Scale bar, 5 µm.

    Techniques Used: Marker

    Trafficking of 4xF M -SNAPtag-NL1 (labelled with JF646) in COS7 cells. 30 min after addition of DDS, NL1 accumulates in a perinuclear GA-like distribution. 120’ after addition of DDS, NL1 is present on the membrane and in vesicular structures located throughout the cell. Addition of thrombin eliminates the surface signal. Scale bar, 10 µm.
    Figure Legend Snippet: Trafficking of 4xF M -SNAPtag-NL1 (labelled with JF646) in COS7 cells. 30 min after addition of DDS, NL1 accumulates in a perinuclear GA-like distribution. 120’ after addition of DDS, NL1 is present on the membrane and in vesicular structures located throughout the cell. Addition of thrombin eliminates the surface signal. Scale bar, 10 µm.

    Techniques Used: Membrane

    ( A ) Live-cell imaging of a cortical neuron expressing 3xF M -GluA1-mCh and TfR-GFP before ER-release (top panels) and 120 min after ER-release (bottom panels). Blue arrowheads indicate locations where GluA1 has redistributed to TfR-GFP positive endosomes. Scale bar, 10 µm. ( B ) Experimental paradigm to visualize individual vesicles trafficking in neurons expressing 3xF M -GluA1-mCh and a green RE marker (either GFP-Rab11 or TfR-GFP). The middle panel shows photobleaching of a dendritic segment (indicated by red square). The right panel is a frame taken 20 s after photobleaching showing endosomes entering the bleached area (blue arrows) Also see and . ( C ) Representative example of a mobile vesicle (orange arrows) associated with both Rab11 and 3xF M -GluA1. Individual frames are taken from 4 Hz dual-color imaging of a dendritic segment following photobleaching. Scale bar, 5 µm. ( D ) Kymographs of dendritic segments following photobleaching showing the movement of Rab11 and GluA1 vesicles at various times following 3xF M -mCh-GluA1 release from the ER. The amount of time elapsed between addition of DDS and imaging is indicated above the kymographs. The black arrow indicates the time of photobleaching. Blue arrowheads denote double-positive mobile vesicles. ( E ) Quantification of the percentage of GluA1 vesicles that also contain the indicated RE marker. The orange bar (+Thr) indicates the inclusion of 1 U/ml thrombin along with DDS. (mean ± SEM, n = 6–8 neurons/timepoint/marker from 3 experiments per marker; 2 independent experiments for thrombin n.s. p=0.31 by unpaired two-tailed Student’s t-test). Numbers on each bar indicate the raw number of double-positive vesicles/total GluA1 vesicles. ( F ) Schematic of soluble anterograde trafficking marker (4xF M -mCh). During vesicle exocytosis, the soluble marker is released from the cell and therefore cannot be recycled. ( G ) Kymographs showing cotrafficking between anterograde soluble marker (4xF M -mCh) and GFP-Rab11 within a segment of dendrite 150 min after DDS addition. Blue arrowheads highlight individual cotrafficking vesicles. ( H ) Quantification of the percentage of vesicles cotrafficking Rab11 and 4xF M -mCh before and 150 min after ER-release (mean ± SEM, no rel. n = 5 neurons; 150 minutes n = 8 neurons from 2 experiments). ( I ) Quantification of cotrafficking between NL1 and Rab11. Numbers on bars indicate raw numbers of vesicles positive for both markers divided by the total number positive for NL1 (mean ± SEM, n = 5–6 neurons/condition from 2 experiments). ( J ) GluA1 accumulates in a subset of spine endosomes following ER release. Shown is a stretch of dendrite from a neuron expressing 3xF M -mCh-GluA1 along with TfR-HaloTag labeled with JF646 before and 150 min following addition of DDS. Orange arrowheads denote spines containing REs. Blue arrows indicate accumulation of GluA1 in spine-resident REs. Dotted yellow outline drawn based on GFP cell fill (not shown). ( K ) Images are of a neuron coexpressing 3xF M -mCh-GluA1 and TfR-HaloTag (JF646) fixed 150 min after addition of DDS in the presence of thrombin (1 U/ml) to prevent visualization of recycled proteins. As in F, blue arrows highlight spine REs that contain trafficking GluA1. Orange arrowheads indicate GluA1-negative spine REs. The right panel is the quantification of the percentage of spines with REs that also contain GluA1 before and 150’ after release in the presence of thrombin. (mean ± SEM, n = 5 neurons/condition from 2 experiments, **p=0.006 unpaired two-tailed Student’s t-test.) Scale bar, 2 μm.
    Figure Legend Snippet: ( A ) Live-cell imaging of a cortical neuron expressing 3xF M -GluA1-mCh and TfR-GFP before ER-release (top panels) and 120 min after ER-release (bottom panels). Blue arrowheads indicate locations where GluA1 has redistributed to TfR-GFP positive endosomes. Scale bar, 10 µm. ( B ) Experimental paradigm to visualize individual vesicles trafficking in neurons expressing 3xF M -GluA1-mCh and a green RE marker (either GFP-Rab11 or TfR-GFP). The middle panel shows photobleaching of a dendritic segment (indicated by red square). The right panel is a frame taken 20 s after photobleaching showing endosomes entering the bleached area (blue arrows) Also see and . ( C ) Representative example of a mobile vesicle (orange arrows) associated with both Rab11 and 3xF M -GluA1. Individual frames are taken from 4 Hz dual-color imaging of a dendritic segment following photobleaching. Scale bar, 5 µm. ( D ) Kymographs of dendritic segments following photobleaching showing the movement of Rab11 and GluA1 vesicles at various times following 3xF M -mCh-GluA1 release from the ER. The amount of time elapsed between addition of DDS and imaging is indicated above the kymographs. The black arrow indicates the time of photobleaching. Blue arrowheads denote double-positive mobile vesicles. ( E ) Quantification of the percentage of GluA1 vesicles that also contain the indicated RE marker. The orange bar (+Thr) indicates the inclusion of 1 U/ml thrombin along with DDS. (mean ± SEM, n = 6–8 neurons/timepoint/marker from 3 experiments per marker; 2 independent experiments for thrombin n.s. p=0.31 by unpaired two-tailed Student’s t-test). Numbers on each bar indicate the raw number of double-positive vesicles/total GluA1 vesicles. ( F ) Schematic of soluble anterograde trafficking marker (4xF M -mCh). During vesicle exocytosis, the soluble marker is released from the cell and therefore cannot be recycled. ( G ) Kymographs showing cotrafficking between anterograde soluble marker (4xF M -mCh) and GFP-Rab11 within a segment of dendrite 150 min after DDS addition. Blue arrowheads highlight individual cotrafficking vesicles. ( H ) Quantification of the percentage of vesicles cotrafficking Rab11 and 4xF M -mCh before and 150 min after ER-release (mean ± SEM, no rel. n = 5 neurons; 150 minutes n = 8 neurons from 2 experiments). ( I ) Quantification of cotrafficking between NL1 and Rab11. Numbers on bars indicate raw numbers of vesicles positive for both markers divided by the total number positive for NL1 (mean ± SEM, n = 5–6 neurons/condition from 2 experiments). ( J ) GluA1 accumulates in a subset of spine endosomes following ER release. Shown is a stretch of dendrite from a neuron expressing 3xF M -mCh-GluA1 along with TfR-HaloTag labeled with JF646 before and 150 min following addition of DDS. Orange arrowheads denote spines containing REs. Blue arrows indicate accumulation of GluA1 in spine-resident REs. Dotted yellow outline drawn based on GFP cell fill (not shown). ( K ) Images are of a neuron coexpressing 3xF M -mCh-GluA1 and TfR-HaloTag (JF646) fixed 150 min after addition of DDS in the presence of thrombin (1 U/ml) to prevent visualization of recycled proteins. As in F, blue arrows highlight spine REs that contain trafficking GluA1. Orange arrowheads indicate GluA1-negative spine REs. The right panel is the quantification of the percentage of spines with REs that also contain GluA1 before and 150’ after release in the presence of thrombin. (mean ± SEM, n = 5 neurons/condition from 2 experiments, **p=0.006 unpaired two-tailed Student’s t-test.) Scale bar, 2 μm.

    Techniques Used: Live Cell Imaging, Expressing, Marker, Imaging, Two Tailed Test, Labeling

    Surface delivery of VSV-G-YFP-4xF M (left) and 4xF M -mCh-NL1 (right) was measured in dissociated cortical neurons after various combinations of temperature and BFA treatment. Experimental conditions are indicated under the bars and are the same as in . Surface intensity is expressed as a percentage of control (37 ˚C, no BFA) surface delivery. Values are reported as mean relative to control ± SEM (n = 5–8 cells/condition, n.s. = not significant by one way ANOVA/Bonferonni post hoc test). Both NL1 and VSV-G experiments were independently repeated with similar results.
    Figure Legend Snippet: Surface delivery of VSV-G-YFP-4xF M (left) and 4xF M -mCh-NL1 (right) was measured in dissociated cortical neurons after various combinations of temperature and BFA treatment. Experimental conditions are indicated under the bars and are the same as in . Surface intensity is expressed as a percentage of control (37 ˚C, no BFA) surface delivery. Values are reported as mean relative to control ± SEM (n = 5–8 cells/condition, n.s. = not significant by one way ANOVA/Bonferonni post hoc test). Both NL1 and VSV-G experiments were independently repeated with similar results.

    Techniques Used: Control


    Figure Legend Snippet:

    Techniques Used: Transfection, Construct, Generated, Derivative Assay, Plasmid Preparation, Western Blot, Recombinant


    Figure Legend Snippet:

    Techniques Used: Construct



    Similar Products

    nl1 orf  (Addgene inc)
    93
    Addgene inc nl1 orf
    ( A ) Schematic depicting inducible release constructs. Multiple copies of self-associating F M domains were fused to target proteins downstream of an ER signal peptide and upstream of a fluorescent protein (FP). DDS dissociates F M domains allowing synchronous ER exit. A furin cleavage site allows removal of the F M domains as they transit the GA. A thrombin cleavage site was included in some constructs so that the FP could be selectively removed from proteins localized at the PM. ( B ) Comparison of 3xF M -mEOS-GluA1 with the endogenous ER-marker BiP before release (left panel) and the Golgi marker GM130 1 hr after DDS addition (right panel). ( C ) Detection of GluA1 surface delivery at various time points following addition of DDS by surface labelling against the extracellular HA-tag of 3xF M -GluA1. ( D ) Quantification of GluA1 surface delivery shown in C (mean ± SEM, n = 10–12 neurons/timepoint from 2 independent experiments). All values normalized to neurons that were not treated with DDS. ( E ) Time-course of <t>NL1</t> surface delivery (mean ± SEM, n = 9–10 neurons/timepoint from 2 independent experiments). ( F ) Localization of surface GluA1 after ER-release. Images taken from insets in panel C. Scale bar, 2 µm.
    Nl1 Orf, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/nl1 orf/product/Addgene inc
    Average 93 stars, based on 1 article reviews
    nl1 orf - by Bioz Stars, 2026-03
    93/100 stars
    		  Buy from Supplier

    Image Search Results


    ( A ) Schematic depicting inducible release constructs. Multiple copies of self-associating F M domains were fused to target proteins downstream of an ER signal peptide and upstream of a fluorescent protein (FP). DDS dissociates F M domains allowing synchronous ER exit. A furin cleavage site allows removal of the F M domains as they transit the GA. A thrombin cleavage site was included in some constructs so that the FP could be selectively removed from proteins localized at the PM. ( B ) Comparison of 3xF M -mEOS-GluA1 with the endogenous ER-marker BiP before release (left panel) and the Golgi marker GM130 1 hr after DDS addition (right panel). ( C ) Detection of GluA1 surface delivery at various time points following addition of DDS by surface labelling against the extracellular HA-tag of 3xF M -GluA1. ( D ) Quantification of GluA1 surface delivery shown in C (mean ± SEM, n = 10–12 neurons/timepoint from 2 independent experiments). All values normalized to neurons that were not treated with DDS. ( E ) Time-course of NL1 surface delivery (mean ± SEM, n = 9–10 neurons/timepoint from 2 independent experiments). ( F ) Localization of surface GluA1 after ER-release. Images taken from insets in panel C. Scale bar, 2 µm.

    Journal: eLife

    Article Title: Golgi-independent secretory trafficking through recycling endosomes in neuronal dendrites and spines

    doi: 10.7554/eLife.27362

    Figure Lengend Snippet: ( A ) Schematic depicting inducible release constructs. Multiple copies of self-associating F M domains were fused to target proteins downstream of an ER signal peptide and upstream of a fluorescent protein (FP). DDS dissociates F M domains allowing synchronous ER exit. A furin cleavage site allows removal of the F M domains as they transit the GA. A thrombin cleavage site was included in some constructs so that the FP could be selectively removed from proteins localized at the PM. ( B ) Comparison of 3xF M -mEOS-GluA1 with the endogenous ER-marker BiP before release (left panel) and the Golgi marker GM130 1 hr after DDS addition (right panel). ( C ) Detection of GluA1 surface delivery at various time points following addition of DDS by surface labelling against the extracellular HA-tag of 3xF M -GluA1. ( D ) Quantification of GluA1 surface delivery shown in C (mean ± SEM, n = 10–12 neurons/timepoint from 2 independent experiments). All values normalized to neurons that were not treated with DDS. ( E ) Time-course of NL1 surface delivery (mean ± SEM, n = 9–10 neurons/timepoint from 2 independent experiments). ( F ) Localization of surface GluA1 after ER-release. Images taken from insets in panel C. Scale bar, 2 µm.

    Article Snippet: 4xF M -NL1 was generated by insertion of NL1 ORF (Addgene #15260, a gift from Peter Schieffelle) into a construct containing 4xF M. This fusion was then subcloned into a pCAG backbone.

    Techniques: Construct, Comparison, Marker

    ER-retained 4xF M -SEP-NL1 coexpressed with an engineered ER-marker TfR-mCh-KDEL in a COS7 cell. Scale bar, 5 µm.

    Journal: eLife

    Article Title: Golgi-independent secretory trafficking through recycling endosomes in neuronal dendrites and spines

    doi: 10.7554/eLife.27362

    Figure Lengend Snippet: ER-retained 4xF M -SEP-NL1 coexpressed with an engineered ER-marker TfR-mCh-KDEL in a COS7 cell. Scale bar, 5 µm.

    Article Snippet: 4xF M -NL1 was generated by insertion of NL1 ORF (Addgene #15260, a gift from Peter Schieffelle) into a construct containing 4xF M. This fusion was then subcloned into a pCAG backbone.

    Techniques: Marker

    Trafficking of 4xF M -SNAPtag-NL1 (labelled with JF646) in COS7 cells. 30 min after addition of DDS, NL1 accumulates in a perinuclear GA-like distribution. 120’ after addition of DDS, NL1 is present on the membrane and in vesicular structures located throughout the cell. Addition of thrombin eliminates the surface signal. Scale bar, 10 µm.

    Journal: eLife

    Article Title: Golgi-independent secretory trafficking through recycling endosomes in neuronal dendrites and spines

    doi: 10.7554/eLife.27362

    Figure Lengend Snippet: Trafficking of 4xF M -SNAPtag-NL1 (labelled with JF646) in COS7 cells. 30 min after addition of DDS, NL1 accumulates in a perinuclear GA-like distribution. 120’ after addition of DDS, NL1 is present on the membrane and in vesicular structures located throughout the cell. Addition of thrombin eliminates the surface signal. Scale bar, 10 µm.

    Article Snippet: 4xF M -NL1 was generated by insertion of NL1 ORF (Addgene #15260, a gift from Peter Schieffelle) into a construct containing 4xF M. This fusion was then subcloned into a pCAG backbone.

    Techniques: Membrane

    ( A ) Live-cell imaging of a cortical neuron expressing 3xF M -GluA1-mCh and TfR-GFP before ER-release (top panels) and 120 min after ER-release (bottom panels). Blue arrowheads indicate locations where GluA1 has redistributed to TfR-GFP positive endosomes. Scale bar, 10 µm. ( B ) Experimental paradigm to visualize individual vesicles trafficking in neurons expressing 3xF M -GluA1-mCh and a green RE marker (either GFP-Rab11 or TfR-GFP). The middle panel shows photobleaching of a dendritic segment (indicated by red square). The right panel is a frame taken 20 s after photobleaching showing endosomes entering the bleached area (blue arrows) Also see and . ( C ) Representative example of a mobile vesicle (orange arrows) associated with both Rab11 and 3xF M -GluA1. Individual frames are taken from 4 Hz dual-color imaging of a dendritic segment following photobleaching. Scale bar, 5 µm. ( D ) Kymographs of dendritic segments following photobleaching showing the movement of Rab11 and GluA1 vesicles at various times following 3xF M -mCh-GluA1 release from the ER. The amount of time elapsed between addition of DDS and imaging is indicated above the kymographs. The black arrow indicates the time of photobleaching. Blue arrowheads denote double-positive mobile vesicles. ( E ) Quantification of the percentage of GluA1 vesicles that also contain the indicated RE marker. The orange bar (+Thr) indicates the inclusion of 1 U/ml thrombin along with DDS. (mean ± SEM, n = 6–8 neurons/timepoint/marker from 3 experiments per marker; 2 independent experiments for thrombin n.s. p=0.31 by unpaired two-tailed Student’s t-test). Numbers on each bar indicate the raw number of double-positive vesicles/total GluA1 vesicles. ( F ) Schematic of soluble anterograde trafficking marker (4xF M -mCh). During vesicle exocytosis, the soluble marker is released from the cell and therefore cannot be recycled. ( G ) Kymographs showing cotrafficking between anterograde soluble marker (4xF M -mCh) and GFP-Rab11 within a segment of dendrite 150 min after DDS addition. Blue arrowheads highlight individual cotrafficking vesicles. ( H ) Quantification of the percentage of vesicles cotrafficking Rab11 and 4xF M -mCh before and 150 min after ER-release (mean ± SEM, no rel. n = 5 neurons; 150 minutes n = 8 neurons from 2 experiments). ( I ) Quantification of cotrafficking between NL1 and Rab11. Numbers on bars indicate raw numbers of vesicles positive for both markers divided by the total number positive for NL1 (mean ± SEM, n = 5–6 neurons/condition from 2 experiments). ( J ) GluA1 accumulates in a subset of spine endosomes following ER release. Shown is a stretch of dendrite from a neuron expressing 3xF M -mCh-GluA1 along with TfR-HaloTag labeled with JF646 before and 150 min following addition of DDS. Orange arrowheads denote spines containing REs. Blue arrows indicate accumulation of GluA1 in spine-resident REs. Dotted yellow outline drawn based on GFP cell fill (not shown). ( K ) Images are of a neuron coexpressing 3xF M -mCh-GluA1 and TfR-HaloTag (JF646) fixed 150 min after addition of DDS in the presence of thrombin (1 U/ml) to prevent visualization of recycled proteins. As in F, blue arrows highlight spine REs that contain trafficking GluA1. Orange arrowheads indicate GluA1-negative spine REs. The right panel is the quantification of the percentage of spines with REs that also contain GluA1 before and 150’ after release in the presence of thrombin. (mean ± SEM, n = 5 neurons/condition from 2 experiments, **p=0.006 unpaired two-tailed Student’s t-test.) Scale bar, 2 μm.

    Journal: eLife

    Article Title: Golgi-independent secretory trafficking through recycling endosomes in neuronal dendrites and spines

    doi: 10.7554/eLife.27362

    Figure Lengend Snippet: ( A ) Live-cell imaging of a cortical neuron expressing 3xF M -GluA1-mCh and TfR-GFP before ER-release (top panels) and 120 min after ER-release (bottom panels). Blue arrowheads indicate locations where GluA1 has redistributed to TfR-GFP positive endosomes. Scale bar, 10 µm. ( B ) Experimental paradigm to visualize individual vesicles trafficking in neurons expressing 3xF M -GluA1-mCh and a green RE marker (either GFP-Rab11 or TfR-GFP). The middle panel shows photobleaching of a dendritic segment (indicated by red square). The right panel is a frame taken 20 s after photobleaching showing endosomes entering the bleached area (blue arrows) Also see and . ( C ) Representative example of a mobile vesicle (orange arrows) associated with both Rab11 and 3xF M -GluA1. Individual frames are taken from 4 Hz dual-color imaging of a dendritic segment following photobleaching. Scale bar, 5 µm. ( D ) Kymographs of dendritic segments following photobleaching showing the movement of Rab11 and GluA1 vesicles at various times following 3xF M -mCh-GluA1 release from the ER. The amount of time elapsed between addition of DDS and imaging is indicated above the kymographs. The black arrow indicates the time of photobleaching. Blue arrowheads denote double-positive mobile vesicles. ( E ) Quantification of the percentage of GluA1 vesicles that also contain the indicated RE marker. The orange bar (+Thr) indicates the inclusion of 1 U/ml thrombin along with DDS. (mean ± SEM, n = 6–8 neurons/timepoint/marker from 3 experiments per marker; 2 independent experiments for thrombin n.s. p=0.31 by unpaired two-tailed Student’s t-test). Numbers on each bar indicate the raw number of double-positive vesicles/total GluA1 vesicles. ( F ) Schematic of soluble anterograde trafficking marker (4xF M -mCh). During vesicle exocytosis, the soluble marker is released from the cell and therefore cannot be recycled. ( G ) Kymographs showing cotrafficking between anterograde soluble marker (4xF M -mCh) and GFP-Rab11 within a segment of dendrite 150 min after DDS addition. Blue arrowheads highlight individual cotrafficking vesicles. ( H ) Quantification of the percentage of vesicles cotrafficking Rab11 and 4xF M -mCh before and 150 min after ER-release (mean ± SEM, no rel. n = 5 neurons; 150 minutes n = 8 neurons from 2 experiments). ( I ) Quantification of cotrafficking between NL1 and Rab11. Numbers on bars indicate raw numbers of vesicles positive for both markers divided by the total number positive for NL1 (mean ± SEM, n = 5–6 neurons/condition from 2 experiments). ( J ) GluA1 accumulates in a subset of spine endosomes following ER release. Shown is a stretch of dendrite from a neuron expressing 3xF M -mCh-GluA1 along with TfR-HaloTag labeled with JF646 before and 150 min following addition of DDS. Orange arrowheads denote spines containing REs. Blue arrows indicate accumulation of GluA1 in spine-resident REs. Dotted yellow outline drawn based on GFP cell fill (not shown). ( K ) Images are of a neuron coexpressing 3xF M -mCh-GluA1 and TfR-HaloTag (JF646) fixed 150 min after addition of DDS in the presence of thrombin (1 U/ml) to prevent visualization of recycled proteins. As in F, blue arrows highlight spine REs that contain trafficking GluA1. Orange arrowheads indicate GluA1-negative spine REs. The right panel is the quantification of the percentage of spines with REs that also contain GluA1 before and 150’ after release in the presence of thrombin. (mean ± SEM, n = 5 neurons/condition from 2 experiments, **p=0.006 unpaired two-tailed Student’s t-test.) Scale bar, 2 μm.

    Article Snippet: 4xF M -NL1 was generated by insertion of NL1 ORF (Addgene #15260, a gift from Peter Schieffelle) into a construct containing 4xF M. This fusion was then subcloned into a pCAG backbone.

    Techniques: Live Cell Imaging, Expressing, Marker, Imaging, Two Tailed Test, Labeling

    Surface delivery of VSV-G-YFP-4xF M (left) and 4xF M -mCh-NL1 (right) was measured in dissociated cortical neurons after various combinations of temperature and BFA treatment. Experimental conditions are indicated under the bars and are the same as in . Surface intensity is expressed as a percentage of control (37 ˚C, no BFA) surface delivery. Values are reported as mean relative to control ± SEM (n = 5–8 cells/condition, n.s. = not significant by one way ANOVA/Bonferonni post hoc test). Both NL1 and VSV-G experiments were independently repeated with similar results.

    Journal: eLife

    Article Title: Golgi-independent secretory trafficking through recycling endosomes in neuronal dendrites and spines

    doi: 10.7554/eLife.27362

    Figure Lengend Snippet: Surface delivery of VSV-G-YFP-4xF M (left) and 4xF M -mCh-NL1 (right) was measured in dissociated cortical neurons after various combinations of temperature and BFA treatment. Experimental conditions are indicated under the bars and are the same as in . Surface intensity is expressed as a percentage of control (37 ˚C, no BFA) surface delivery. Values are reported as mean relative to control ± SEM (n = 5–8 cells/condition, n.s. = not significant by one way ANOVA/Bonferonni post hoc test). Both NL1 and VSV-G experiments were independently repeated with similar results.

    Article Snippet: 4xF M -NL1 was generated by insertion of NL1 ORF (Addgene #15260, a gift from Peter Schieffelle) into a construct containing 4xF M. This fusion was then subcloned into a pCAG backbone.

    Techniques: Control

    Journal: eLife

    Article Title: Golgi-independent secretory trafficking through recycling endosomes in neuronal dendrites and spines

    doi: 10.7554/eLife.27362

    Figure Lengend Snippet:

    Article Snippet: 4xF M -NL1 was generated by insertion of NL1 ORF (Addgene #15260, a gift from Peter Schieffelle) into a construct containing 4xF M. This fusion was then subcloned into a pCAG backbone.

    Techniques: Transfection, Construct, Generated, Derivative Assay, Plasmid Preparation, Western Blot, Recombinant

    Journal: eLife

    Article Title: Golgi-independent secretory trafficking through recycling endosomes in neuronal dendrites and spines

    doi: 10.7554/eLife.27362

    Figure Lengend Snippet:

    Article Snippet: 4xF M -NL1 was generated by insertion of NL1 ORF (Addgene #15260, a gift from Peter Schieffelle) into a construct containing 4xF M. This fusion was then subcloned into a pCAG backbone.

    Techniques: Construct